'Genotyping Microarray for CSNB-Associated Genes'

Zeitz, C; Labs, S; Lorenz, B; Forster, U; Üksti, J; Kroes, H Y; De Baere, E; Leroy, B P; Cremers, F P M; Wittmer, M; van Genderen, M M; Sahel, J A; Audo, I; Poloschek, C M; Mohand-Said, S; Fleischhauer, J C; Hüffmeier, U; Moskova-Doumanova, V; Levin, A V; Hamel, C P; Leifert, D; Munier, F L; Schorderet, D F; Zrenner, E; Friedburg, C; Wissinger, B; Kohl, S; Berger, W (2009). Genotyping Microarray for CSNB-Associated Genes. Investigative Ophthalmology and Visual Science, 12(50):5919-5926. University of Zurich Postprint available at: Zurich Open Repository and Archive http://www.zora.uzh.ch Posted at the Zurich Open Repository and Archive, University of Zurich. http://www.zora.uzh.ch

Winterthurerstr. 190 Originally published at: CH-8057 Zurich Investigative Ophthalmology and Visual Science 2009, 12(50):5919-5926. http://www.zora.uzh.ch

Year: 2009

Genotyping Microarray for CSNB-Associated Genes

Posted at the Zurich Open Repository and Archive, University of Zurich. http://www.zora.uzh.ch

Originally published at: Investigative Ophthalmology and Visual Science 2009, 12(50):5919-5926. Genotyping Microarray for CSNB-Associated Genes

Abstract

PURPOSE. Congenital stationary night blindness (CSNB) is a clinically and genetically heterogeneous retinal disease. Although electroretinographic (ERG) measurements can discriminate clinical subgroups, the identification of the underlying genetic defects has been complicated for CSNB because ofgenetic heterogeneity, the uncertainty about the mode of inheritance, and time-consuming and costly mutation scanning and direct sequencing approaches. METHODS. To overcome these challenges and to generate a time- and cost-efficient mutation screening tool, the authors developed a CSNB genotyping microarray with arrayed primer extension (APEX) technology. To cover as many mutations as possible, a comprehensive literature search was performed, and DNA samples from a cohort of patients with CSNB were first sequenced directly in known CSNB genes. Subsequently, oligonucleotides were designed representing 126 sequence variations in RHO, CABP4, CACNA1F, CACNA2D4, GNAT1,GRM6, NYX, PDE6B, and SAG and spotted on the chip. RESULTS. Direct sequencing of genes known to be associated with CSNB in the study cohort revealed 21 mutations (12 novel and 9 previously reported). The resultant microarray containing oligonucleotides, which allow to detect 126 known and novel mutations, was 100% effective in determining the expected sequence changes in all known samples assessed. In addition, investigation of 34 patients with CSNB who were previously not genotyped revealed sequence variants in 18%, of which 15% are thought to be disease-causing mutations. CONCLUSIONS. This relatively inexpensive first-pass genetic testing device for patients with a diagnosis of CSNB will improve molecular diagnostics and genetic counseling of patients and their families and gives the opportunity to analyze whether, for example, more progressive disorders such as cone or cone-rod dystrophies underlie the same gene defects. Genotyping Microarray for CSNB-Associated Genes

Christina Zeitz,1,2,3 Stephan Labs,1 Birgit Lorenz,4 Ursula Forster,1 Janne U¨ksti,5 Hester Y. Kroes,6 Elfride De Baere,7 Bart P. Leroy,7,8 Frans P. M. Cremers,9,10 Mariana Wittmer,1 Maria M. van Genderen,11 Jose´-Alain Sahel,2,3,12 Isabelle Audo,2,3,12 Charlotte M. Poloschek,13 Saddek Mohand-Saïd,12 Johannes C. Fleischhauer,14 Ulrike Hu¨ffmeier,15 Veselina Moskova-Doumanova,2,3 Alex V. Levin,16 Christian P. Hamel,17 Dorothee Leifert,18 Francis L. Munier,19 Daniel F. Schorderet,20 Eberhart Zrenner,21 Christoph Friedburg,4 Bernd Wissinger,22 Susanne Kohl,22 and Wolfgang Berger1

PURPOSE. Congenital stationary night blindness (CSNB) is a though electroretinographic (ERG) measurements can discrim- clinically and genetically heterogeneous retinal disease. Al- inate clinical subgroups, the identiﬁcation of the underlying genetic defects has been complicated for CSNB because of genetic heterogeneity, the uncertainty about the mode of in-

1 heritance, and time-consuming and costly mutation scanning From the Division of Medical Molecular Genetics and Gene Diag- and direct sequencing approaches. nostics, Institute of Medical Genetics, University of Zurich, Zurich, Swit- zerland; 2INSERM (Institut National de la Sante´ et de la Recherche Me´di- METHODS. To overcome these challenges and to generate a cale), UMR_S968, and the 3Department of Genetics, Institut de la Vision, time- and cost-efficient mutation screening tool, the authors Universite´ Pierre et Marie Curie (UPMC), Universite´ Paris 06, Paris, France; developed a CSNB genotyping microarray with arrayed primer the 4Department of Ophthalmology, Justus-Liebig-University Giessen, Uni- extension (APEX) technology. To cover as many mutations as versita¨tsklinikum Giessen and Marburg, Giessen Campus, Giessen, Ger- 5 6 possible, a comprehensive literature search was performed, many; Asper Biotech, Tartu, Estonia; the Department of Medical Genet- and DNA samples from a cohort of patients with CSNB were ics, University Medical Center Utrecht, Utrecht, The Netherlands; the 7Center for Medical Genetics and 8Department of Ophthalmology, Ghent first sequenced directly in known CSNB genes. Subsequently, University Hospital, Ghent, Belgium; the 9Department of Human Genetics, oligonucleotides were designed representing 126 sequence Radboud University Nijmegen Medical Centre, Nijmegen, The Nether- variations in RHO, CABP4, CACNA1F, CACNA2D4, GNAT1, lands; the 10Nijmegen Centre for Molecular Life Sciences, Radboud Uni- GRM6, NYX, PDE6B, and SAG and spotted on the chip. versity Nijmegen, Nijmegen, The Netherlands; the 11Institute for the 12 RESULTS. Direct sequencing of genes known to be associated Visually Impaired, Zeist, The Netherlands; INSERM CIC 503, Centre with CSNB in the study cohort revealed 21 mutations (12 novel Hospitalier National d’Ophtalmologie des Quinze-Vingts, Paris, France; the 13Department of Ophthalmology, University of Freiburg, Freiburg, Ger- and 9 previously reported). The resultant microarray contain- many; the 14Department of Ophthalmology, University Hospital Bern, ing oligonucleotides, which allow to detect 126 known and Bern, Switzerland; the 15Institute for Human Genetics, University Erlan- novel mutations, was 100% effective in determining the ex- gen, Germany; the 16Pediatric Ophthalmology and Ocular Genetics, Wills pected sequence changes in all known samples assessed. In Eye Institute, Philadelphia, Pennsylvania; 17INSERM U 583, Physiopatholo- addition, investigation of 34 patients with CSNB who were gie et The´rapie des De´ficits Sensoriels et Moteurs, Institut des Neuro- previously not genotyped revealed sequence variants in 18%, sciences de Montpellier, Hoˆpital Saint-Eloi, Montpellier, France; the 18De- of which 15% are thought to be disease-causing mutations. partment of Ophthalmology, University Hospital Basel, Basel, Switzerland; the 19Unit of Oculogenetics, Jules Gonin Eye Hospital, Lausanne, Switzer- CONCLUSIONS. This relatively inexpensive first-pass genetic test- land; the 20Institut de Recherche en Ophtalmologie (IRO), Ecole Polytech- ing device for patients with a diagnosis of CSNB will improve nique Fe´de´rale de Lausanne, University of Lausanne, Sion, Switzerland; molecular diagnostics and genetic counseling of patients and 21The University Eye Clinic and the 22Molecular Genetics Laboratory, their families and gives the opportunity to analyze whether, for Institute for Ophthalmic Research, Centre for Ophthalmology, University example, more progressive disorders such as cone or cone–rod Clinics Tu¨bingen, Tu¨bingen, Germany. dystrophies underlie the same gene defects. (Invest Ophthal- Supported by Forschungeskredit, University of Zurich (CZ), Foun- mol Vis Sci. 2009;50:5919–5926) DOI:10.1167/iovs.09-3548 dation Voir et Entendre (CZ), BQR, UPMC, Universite´ Paris 06 (CZ), DFG Grant ZR1/17-2/KFO 134 (EZ, BW, SK), Swiss National Science Foundation Grant 32-111948/1 (FLM), the Research Foundation ongenital stationary night blindness (CSNB) is a clinically Flanders Grant G.0043.06N (BPL, EDB), and a grant from The Founda- Cand genetically heterogeneous retinal disease. It can be tion Fighting Blindness (IA, SM-S, J-AS). associated with deficiency of vision under dim light conditions, Submitted for publication February 10, 2009; revised April 29 and nystagmus, refractive error, or retinal changes. Electroretinog- June 4, 2009; accepted August 26, 2009. Disclosure: C. Zeitz, None; S. Labs, None; B. Lorenz, None; U. raphy is helpful in confirming and subclassifying the disorder. Forster, None; J. U¨ ksti, None; H.Y. Kroes, None; E. De Baere, None; The disease can also be classified with respect to the gene B.P. Leroy, None; F.P.M. Cremers, None; M. Wittmer, None; M.M. defect. Mutations in genes involved in the phototransduction van Genderen, None; J.-A. Sahel, None; I. Audo, None; C.M. Po- cascade (GNAT1, PDE6B, RHO, RHOK, and SAG) are among loschek, None; S. Mohand-Saïd, None; J.C. Fleischhauer, None; U. those that can lead to autosomal dominant CSNB. Although the Hu¨ffmeier, None; V. Moskova-Doumanova, None; A.V. Levin, phenotype of patients with mutations in GNAT1, PDE6B,or None; C.P. Hamel, None; D. Leifert, None; F.L. Munier, None; D.F. RHO may vary, the disease course seems to be stationary with Schorderet, None; E. Zrenner, None; C. Friedburg, None; B. Wiss- primarily scotopic vision affected.1 Mutations in RHOK and inger, None; S. Kohl, None; W. Berger, None SAG lead to Oguchi disease,2 which is a rare, autosomal reces- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- sive, nonprogressive congenital night blindness. It is character- ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. ized by a diffuse grayish white discoloration of the fundus that Corresponding author: Christina Zeitz, Institut de la Vision, De- disappears after a long period of dark adaptation (Mizuo phe- 3 partment of Genetics, Team 4, 17, Rue Moreau, 75012 Paris, France; nomenon). Reduced rod function will improve after an ex- [email protected]. tended period of dark adaptation. Mutations in genes involved

Investigative Ophthalmology & Visual Science, December 2009, Vol. 50, No. 12 Copyright © Association for Research in Vision and Ophthalmology 5919 5920 Zeitz et al. IOVS, December 2009, Vol. 50, No. 12 downstream of the phototransduction cascade can lead to request). Since none of our patients showed the typical fundus asso- either a complete or incomplete type of CSNB. Both types are ciated with Oguchi’s disease, no sequence analysis of SAG and RHOK characterized by an absent or severely reduced b-wave in the was performed. mixed ERG response, revealing a so-called electronegative ERG. The incomplete phenotype is characterized by a defect in Design of the CSNB Microarray: APEX Technology the ON/OFF pathway and has been associated with mutations We used APEX technology for designing a new microarray which is in genes (CACNA1F, CABP4, and CACNA2D4) that are essen- able to detect the 126 different CSNB-related variants. The assay is tial for glutamate release from photoreceptors to the adjacent based on single-primer nucleotide extension,12 and subsequently con- bipolar cells. Although mutations in CACNA1F are associated verted to an array format.13 Detailed description of the methodology is with X-linked recessive inheritance, mutations in CABP4 and available (Asper Biotech, Ltd., http://www.asperbio.com) and provided in CACNA2D4 are associated with an autosomal recessive trait. the Supplementary Data, http://www.iovs.org/cgi/content/full/50/12/ The complete CSNB phenotype is mainly associated with a 5919/DC1. In brief, 5Ј-modified (6-amino linker), sequence-specific defect in the ON pathway. It has been associated with a gene oligonucleotides are arrayed on a glass slide. These oligonucleotides important for glutamate uptake (GRM6) and a gene of un- are designed with their 3Ј-end immediately adjacent to the variable known function (NYX). Mutations in GRM6 lead to autosomal site. PCR-prepared and -fragmented target nucleic acids are annealed to recessive CSNB. Alterations in NYX lead to X-linked recessive oligonucleotides on the slide, followed by sequence-specific extension CSNB (reviewed in Zeitz4). Although the disease course of of the 3Ј-ends of primers with fluorescence-labeled nucleotide ana- CSNB has been described as nonprogressive, it may be pro- logues (ddNTPs) by DNA polymerase.14 Reading of the incorporated gressive, at least in some patients carrying mutations in fluorescence identifies the target sequence. APEX and PCR oligonucle- CACNA1F,5–7 CABP4,8 or CACNA2D4.9 In summary, to date, otide primers were designed according to the wild-type gene se- 10 different genes have been associated with CSNB, with the quences (ref. numbers.: CABP4, NM_145200; CACNA1F, AJ006216; majority (80%) of mutations identified in CACNA1F and NYX.4 CACNA2D4, NM_172364; GNAT1, NM_144499; GRM6, NM_000843; It is difficult in some cases to define the appropriate gene NYX, AJ278865; PDE6B, NM_000283; RHO, NM_000539; and SAG, for mutation screening, because clinical data do not clearly NM_000541; provided in the public domain by the National Center for identify the subtype or the mode of inheritance is not obvious Biotechnology Information [NCBI], Bethesda, MD at http://www. (e.g., sporadic cases). In addition, disease-associated patho- snpper.chip.org, http://www.ncbi.nlm.nih.gov) for both sense and genic variants identified in the 10 known genes so far have antisense strands. shown to be substantially heterogeneous with regard to clinical phenotypes. Currently, more than 100 different disease- Databases Used to Predict the Pathogenic associated variants have been identified in known CSNB genes. To generate a time- and cost-efficient mutation-screening Character of a Sequence Alteration tool, we sought to develop a CSNB genotyping microarray. To The following databases were used to evaluate the potential pathoge- cover as many mutations as possible on such a diagnostic tool, nicity of sequence alterations: NCBI: http://www.ncbi.nlm.nih.gov/ the DNA from our cohort of patients with CSNB was first sites/entrez?dbϭsnp&cmdϭsearch&term/; Human Genome Browser: sequenced in known CSNB-associated genes. These samples http://genome.brc.mcw.edu/cgi-bin/hgBlat/; GenCards: http://www. and those from previously characterized patients were also genecards.org/; PolyPhen (Polymorphism Phenotyping, http://genetics. used to validate the microarray. The microarray was further bwh.harvard.edu/pph/, based on the information of sequence ho- tested on DNA samples of patients with CSNB of unknown mologies and mapping of the affected amino acid to known 3-D genotype. protein structures15,16; and SIFT (Sorting Intolerant From Tolerant, http://blocks.fhcrc.org/sift/SIFT.html, Fred Hutchinson Cancer Center, Seattle, WA), which uses sequence homologies to predict whether an METHODS amino acid will affect protein function.17 CSNB mutations are anno- Patients tated according to the recommendation of the Human Genome Varia- tion Society with nucleotide position ϩ1 corresponding to the A of the The patients with CSNB involved in the study had the disease diag- translation initiation codon ATG in the cDNA nomenclature (http:// nosed in different centers in Europe and Canada (The Netherlands: www.hgvs.org/mutnomen). Utrecht, Nijmegen, and Zeist; Belgium: Ghent; France: Paris and Mont- pellier; Germany: Freiburg, Erlangen, Giessen, Regensburg and Tu¨- bingen; Switzerland: Bern, Lausanne, and Basel; and Canada: Toronto). RESULTS Research procedures were conducted in accordance with institutional guidelines and the Declaration of Helsinki. Before genetic testing, Design of the CSNB Genotyping Microarray informed consent was obtained at each site from all patients, for To date, more than 100 CSNB-associated mutations in 10 dif- diagnostic and/or research purposes, as appropriate. ferent genes have been identified (see review by Zeitz4). To Selection of Oligonucleotide Sequences to be cover as many mutations as possible on a genotyping microarray, we first sequenced the samples from our CSNB DNA Spotted on the Microarray cohort in known CSNB genes. We identified 21 different mu- For the arrayed primer extension (APEX) microarray (Asper Biotech tations (Supplementary Table S1; both Supplementary Tables Ltd., Tartu, Estonia), 126 sequence variants were selected from multi- are online at http://www.iovs.org/cgi/content/full/50/12/5919/ ple sources, including recent mutations identified in our laboratory and DC1). These included two recently described mutations: a mutations or putative polymorphisms found in a comprehensive liter- RHO mutation that co-segregates with an autosomal dominant ature and database search.4 DNA was extracted by standard methods CSNB-phenotype in a large Swiss family and an NYX deletion (detailed information is available on request) and mutation analyses of frequently occurring in Flemish patients with CSNB (Supple- CABP4, CACNA1F, CACNA2D4, GRM6, NYX, and RHO were per- mentary Table S1). In addition, seven known mutations in the formed as described recently.8–11 Mutation analyses for GNAT1 and two X-linked genes were identified: three in NYX and four in PDE6B were performed by PCR-amplification of the 8 coding exons of CACNA1F (Supplementary Table S1). To our knowledge, the GNAT1 in 5 amplicons and the 22 coding exons of PDE6B in 20 remaining 12 mutations have not been described. Six of those fragments, by applying a polymerase enzyme (HotFire, Tartu, Estonia) were found in NYX and six in CACNA1F (Table 1, Supplemen- and subsequently using direct sequencing (detailed conditions on tary Table S1). From those patients in whom ERG examinations IOVS, December 2009, Vol. 50, No. 12 CSNB-Genotyping Microarray 5921

TABLE 1. Summary of Novel CSNB Causing Mutations in NYX discriminated the complete and incomplete form of CSNB, and CACNAIF mutations in NYX were found to be associated in patients with the complete form, whereas mutations in CACNA1F were Nucleotide associated with incomplete CSNB. Some patients with Gene/Exon Change Effect CACNA1F mutations showed a slightly more progressive phe- NYX notype. Segregation of the respective mutation with the phe- 3 c.65GϾA p.Trp22Stop notype was shown for those patients for whom other family 3 c.143GϾA p.Cys48Tyr members were available for analysis (e.g., patient 715.01; Fig. 3 c.187GϾT p.Glu63Stop 1). In addition, control DNA samples were investigated for 3 c.518GϾC p.Arg173Pro novel mutations of uncertain pathogenicity (Supplementary 3 c.607CϾT p.Gln203Stop Table S1). 3 c.1370_1387del18 p.Gln457_Ala463delinsPro These mutations, a large fraction of previously described CACNAIF mutations, and sequence variants with unknown pathogenic 7 c.935delA p.Asp312ThrfsX10 character were used to generate the genotyping microarray. 23 c.2797GϾT p.Asp933Tyr 28 c.3400GϾA p.Glu1134Lys Speciﬁc deletions or mutations that did not reveal a speciﬁc 29 c.3471_3472delGC p.Gln1157HisX25 signal during the validation procedure were omitted. This 38 c.4424TϾC p.Leu1475Pro method resulted in a genotyping microarray containing 126 38 c.4466CϾG p.Pro1481A1a sequence variants of nine different genes implicated in CSNB: 2 CABP4,63CACNA1F,2CACNA2D,1GNAT1,12GRM6,37 Direct sequencing of DNA from patients revealed six novel NYX NYX,1PDE6B,4RHO, and 4 SAG mutations. To facilitate the and six novel CACNA1F mutations.

FIGURE 1. The electropherogram shows the novel dinucleotide deletion c.3471_3472delGC in exon 29 of CACNA1F in patient 715.01. The mother (715.02) was heterozygous for the deletion. The grandfather (715.03) and the brother (715.04) were hemizygous for this mutation. Squares: males; circles: females; dots: carriers; ﬁlled symbols: affected; open symbols: healthy. 5922 Zeitz et al. IOVS, December 2009, Vol. 50, No. 12

TABLE 2. Detection of Known Mutations in Patients with the CSNB Genotype Microarray

Exon Nucleotide Index Phenotype Gene Intron change Effect Publication Interpretation

CH2718 Incomplete CSNB CACNAIF Exon 7 c.945_947delCTT p.Phe316del 21 Disease causing D0706932 Incomplete CSNB CACNAIF Intron 21 c.2673ϩ3GϾA Splice defect 18 SNP or modiﬁer 27538 Incomplete CSNB CACNAIF Exon 24 c.2899CϾT p.Arg967Stop 27, 31 Disease causing C1C00196 Incomplete CSNB CACNAIF Exon 25 c.3019GϾA p.Gly1007Arg 18 Disease causing C1C00748 Incomplete CSNB CACNAIF Exon 33 c.3862CϾT p.Arg1288Stop 21, 33 Disease causing 825.01 Incomplete CSNB CACNAIF Exon 35 c.4091TϾA p.Leu1364His 27 Disease causing

interpretation of the outcome of such a microarray screening member of the family, and he shows a clear incomplete type of with CSNB patient samples with unknown genotype, we pro- CSNB (Table 2). vide the original references in this study (Supplementary Table The 12-year-old male patient CIC00196 with a S2). c.3019GϾA transition (p.Gly1007Arg) substitution in exon 25 of CACNA1F is a sporadic case. Mutation analysis in his Validation of the CSNB Genotyping Microarray father and mother did not show the mutation and thus the Ͼ The genotyping CSNB microarray was first validated with c.3019G A transition represents a de novo mutation. Clin- marked oligonucleotides, which served as positive internal ical data from this patient were suggestive of the incomplete controls. In addition, a negative control (with no DNA) was type of CSNB (Table 2). Ͼ used to investigate the nonspecific background signal. To fur- The 18-year old-male patient CIC00748 with a c.3862C T ther test the capability of the microarray to detect sequence transition in CACNA1F leading to a nonsense mutation alterations, we screened 39 DNAs from patients with 37 (p.Arg1288Stop) in exon 33 mentioned having an affected known variants. All the expected variants were detected with cousin, indicative of an X-linked mode of inheritance. Again, 100% accuracy (Supplementary Table S2). clinical observations were suggestive of the incomplete type of CSNB (Table 2). Screening Results in Previously Untested Patients Clinical data for patient CH2718, revealing the with CSNB c.945_947delCTT (p.Phe316del) in exon 7 in CACNA1F, and of patient 825.01 with a c.4091TϾA mutation in CACNA1F To further evaluate the clinical validity of the CSNB array, we (p.Leu1364His), showed an incomplete type of CSNB (Ta- screened 34 additional patients with CSNB with unknown ble 2). gene defect. Patients from different clinical centers were in- Patient D0706932, with the putative splice site mutation, cluded in this study. This multicenter recruitment resulted in c.2673ϩ3GϾA in intron 21 in CACNA1F represents a simplex variability in the methods used to clinically assess patients male case showing clinically signs and symptoms of incom- included in the study. The screening, which was confirmed by plete CSNB (Table 2). However, further investigation of this direct sequencing, resulted in the detection of six sequence variant explained in the next paragraph showed that this vari- alterations in CACNA1F, of which five are thought to be disease ant is probably not disease causing. causing (Table 2, Supplementary Table S2). In summary, all six patients with a CACNA1F sequence In summary, our screening detected sequence variants in alteration detected by our microarray showed an incomplete 18% of these patients, of which 15% are thought to be patho- CSNB phenotype that is in accordance with this gene defect. genic. Except for the predicted splice site mutation c.2673ϩ3GϾAin intron 21, the identified mutations can be considered to be Rationale for Six CSNB Patients Screened on the disease causing. Microarray Showing a Known CACNA1F Mutation Patient 27538, had a c.2899CϾT mutation in CACNA1F, which Detection of Additional Variants in Patients with is predicted to lead to a premature stop codon at amino acid Known Genotype position 967 (p.Arg967Stop). His parents are consanguineous, and autosomal recessive inheritance was suspected. Because of The microarray screening revealed three additional CACNA1F the mutation identified in CACNA1F the assumed mode of variants in patients with known disease-associated sequence inheritance was shown to be wrong. He is the only affected variations. These variants were verified by direct sequencing

TABLE 3A. Variants Detected by Screening Patients with Known Genotype or Unclear Pathogenic Character

Exon Nucleotide Gene Intron Change Effect Publication Index Interpretation

CACNAIF Exon 13 c.1523GϾA p.Arg508Gln 27 5854, 2422 SNP or modifier CACNAIF Exon 16 c.2204AϾC p.Asn735Thr 8 13276 SNP or modifier, unclear CACNAIF Intron 21 c.2673ϩ3GϾA Splice defect 18 MT, 446.1, DO706932 SNP or modifier CACNAIF Intron 24 c.2938ϩIGϾA Splice defect 21 1344.01 Female SNP or modifier, unclear GRM6 Exon 3 c.727GϾT p.Val243Phe This study, 23 13154, 7330 SNP or modifier GRM6 Exon 3 c.824GϾA p.Gly275Asp This study 8798 Unclear GRM6 Exon 8 c.2090AϾT, p.Gln697Lcu This study 7699 Unclear CACNA2D4 Exon 25 c.2452CϾT p.Arg818Cys This study Not tested Unlcear

Based on the phenotype, functional analysis, or co-segregation studies, these variants are interpreted as nonpathogenic or of unclear pathogenicity. IOVS, December 2009, Vol. 50, No. 12 CSNB-Genotyping Microarray 5923

TABLE 3B. Disease-Associated Genotypes of Patients with Second Variant Listed in Table 3A

Exon Nucleotide Index Phenotype Gene Intron Change Effect Publication Interpretation

5854 CSNB NYX Exon 3 c.647AϾG p.Asn216Ser This study, 22, 23 Disease causing 2422 CSNB NYX Exon 3 c.1040TϾC p.Leu347Pro 26 Disease causing MT Complete arCSNB GRM6 Exon 6 c.1214TϾC p.Ile405Thr 20 Disease causing 446.01 Complete XICSNB NYX Exon 3 c.518GϾC p.Arg173Pro This study Disease causing 13276 Incomplete arCSNB CABP4 Exon 2 c.370CϾT p.Argl24Cys 7 Disease causing Exon 6 c.800_801delAG p.Glu267ValfsX92 7330 CSNB NYX Exon 3 c.607CϾT p.Gln203Stop This study Disease causing

Based on phenotype, functional analysis, or co-segregation studies, these mutations are interpreted as disease causing.

(Supplementary Table S2 and Table 3A) and described in more this sequence variant seems not to be disease causing the site detail in the following sections. is highly conserved and predicted to influence splicing. Splic- -Predicted Splice Site Mutation: c.2673؉3G>A. Patient ing assays to be performed in the future will show the conse MT (a woman) showed a homozygous c.1214TϾC transition quences of this sequence variant. (p.Ile405Thr) in GRM6, and patient 446.1 revealed a c.1523G>A Transition Leading to a p.Arg508Gln. Two c.518GϾC transversion (p.Arg173Pro) in NYX (Table 3B). In patients, 5854 and 2422, showed a c.1523GϾA transition caus- addition, both patients carried a known predicted splice site ing a p.Arg508Gln substitution in CACNA1F (Table 3A), in mutation (c.2673ϩ3GϾA)18 in CACNA1F (Table 3A). Clinical addition to the already identified p.Asn216Ser (patient 5854) examination including electroretinography of the female pa- and p.Leu347Pro exchanges (patient 2422) in NYX, respec- tient MT revealed autosomal recessive complete CSNB.19 Func- tively (Table 3B). Re-evaluation of the clinical records of pa- tional analysis of the c.1214TϾC transition in GRM6 showed tient 5854 revealed no details about the CSNB phenotype. The that the phenotype is due to the absence of the receptor on the mutation p.Asn216Ser in NYX has been described to be disease cell surface.20 The c.2673ϩ3GϾA change in CACNA1F was causing in two independent studies (three families).22,23 In one heterozygous in MT. These findings indicate that the GRM6 of these studies co-segregation was shown in two affected mutation is the disease-causing mutation in this patient and not family members.22 Furthermore, the amino acid asparagine is the CACNA1F variant (Tables 3A, 3B). highly conserved in a leucine-rich repeat.23 These findings Clinical examination including electroretinography of pa- strongly argue for the fact that this sequence variant in NYX is tient 446.1 was consistent with complete XlCSNB. Mutation indeed the disease-causing mutation (Table 3B). analysis in NYX identified a novel hemizygous c.518GϾC trans- Patient 2422 is a member of the large Dutch CSNB family version leading to a p.Arg173Pro substitution, which co-segre- that was used to link CSNB to DXS228, MAOB, and NDP.24 gates with the phenotype (the affected brother and grandfa- Patients in this family showed clinical symptoms of night blind- ther were hemizygous, whereas the mother was ness, but it is also unclear whether they are affected by the heterozygous). The association of the complete form of X- incomplete or complete type of CSNB. Later, this linkage in- linked recessive CSNB with NYX mutations and co-segregation terval was refined to DXS993 and DXS228. Subsequently, the of the mutation in the family supports the hypothesis that the NYX gene was identified in this region and shown to carry a NYX mutation is indeed the disease-causing mutation and not mutation in this family (c.1040TϾC; p.Leu347Pro) and in other the CACNA1F splice site mutation (Tables 3A, 3B). patients.22,25,26 CACNA1F, in contrast was mapped centro- The predicted splice site mutation c.2673ϩ3GϾAin meric to DXS2722 and DXS255. With respect to the haplo- CACNA1F was first described in two patients from two inde- types24 of patient 2422, we suspect that all affected family pendent families (T10, T26).18 T10 was a simplex case and members but also one unaffected male (III-524) carry the thus co-segregation analysis was not performed, whereas co- p.Arg508Gln substitution in CACNA1F in addition to the NYX segregation was observed in the family of T26 (the affected mutation. These findings suggest that, at least for this family brother was also hemizygous and the mother was a carrier). the NYX mutation and not the amino acid substitution in Because of co-segregation and the site of the variant in the CACNA1F is indeed disease causing. We cannot exclude the consensus sequence of the splice donor site, the possibility that the CACNA1F sequence alteration modifies the c.2673ϩ3GϾA was assumed to be pathogenic. However, now phenotype (Tables 3A, 3B). different databases (NCBI, Human Genome Browser) indicate that this substitution represents an SNP (rs41312124), although the frequency in different populations has not been defined. Taking into account the complete phenotype of our patients, as well as results of functional20 and co-segregation analyses, we suggest that the c.2673ϩ3GϾAinCACNA1F re- flects either a rare SNP or a variant modifying the phenotype of the patients (Table 3A). Predicted Splice Site Mutation: c.2938؉1G>A. Another known CACNA1F predicted splice site mutation (c.2938ϩ 1GϾA)21 was heterozygous in case 1344.01. This woman was clinically diagnosed with incomplete CSNB. Co-segregation analysis revealed that her unaffected sister was also heterozygous for the variation, her unaffected father was hemizygous for the variation, and her mother had two wild-type alleles (Fig. FIGURE 2. Segregation analysis of a heterozygous splice site mutation 2). These findings indicate that this sequence alteration did not (c.2938ϩ1GϾA). The index patient (arrow) as well as the unaffected co-segregate with the phenotype and thus, at least in this sister was heterozygous for the variation. The father was hemizygous family, is not disease causing (Table 3A). Despite the fact that for the variation, and the mother had two unaffected alleles. 5924 Zeitz et al. IOVS, December 2009, Vol. 50, No. 12

The c.1523GϾA transition leading to a p.Arg508Gln in aging, whereas in SIFT, it was considered to be benign. A CACNA1F itself was first described by Strom et al.27 in two previously unreported third heterozygous GRM6 sequence patients from two different families (03 and 06) (Table 3A). It variant was found in exon 8 (c.2090AϾT, p.Gln697Leu) in a was excluded in 120 control chromosomes analyzed by SSCP. female patient (7699). Again, due to the absence of a second The index patient of family 06 had a second substitution in mutation, the pathogenic character of the c.2090AϾT substi- CACNA1F (p.Leu849Pro) that was suggested to be non–disease tution is not clear. It was neither published nor predicted as an causing as it “affected a non-conserved leucine.” Hoda et al.28 SNP in the databases available and listed herein. Polyphen and investigated the functional effect of p.Arg508Gln. They found SIFT predicted this variant as probably damaging. However, no changes in the gating properties of the mutant channel because of the absence of a second mutation, it is not clear subunit after heterologous expression in Xenopus laevis oo- whether this substitution in GRM6 is indeed disease causing. cytes, but identified a temperature-dependent altered expres- Similarly, when we screened CACNA2D4 for sequence alter- sion density of the Cav1.4 protein encoded by CACNA1F.It ations in our CSNB cohort, we detected a heterozygous was thus theorized that the amount of expressed protein is c.2452CϾT transition (p.Arg818Cys) in exon 25 in a patient critical for the correct function of the channel.28 Different with incomplete CSNB. A second mutation was not detected, databases are available for use in investigating whether an and thus the pathogenic character remains to be unresolved. It identified variation is an SNP, based on allele frequency and was neither published nor predicted as an SNP in available evolutionary conservation (NCBI, UCSC Human Genome databases. Polyphen and SIFT predicted this variant as proba- Browser and GeneCards). According to several databases, the bly damaging (Table 3A). Functional studies are needed to sequence variation c.1523GϾA (p.Arg508Gln) represents an determine whether these sequence variations are pathogenic. SNP (rs34162630). Moreover, 294 samples have been investigated in populations from North America, Europe, East Asia, Summary of CSNB Microarray and West Africa and the A was found at a frequency of 0.25 (04.11.2008). Together, these findings indicate that the In total of 126 sequence variants can be detected by the CSNB c.1532GϾA transition in CACNA1F is either a polymorphism or microarray. Based on the literature and our own validation of a sequence alteration modifying the phenotype (Table 3A). some cases, 118 of those are disease causing, whereas 8 of them are of uncertain pathogenic character, representing SNPs or modifiers (Table 3B, Supplementary Table S2). The microar- Putative Polymorphisms and/or Disease- ray was 100% effective in detecting known variants and re- Modifying Sequence Variations on the vealed a sequence variant in 18%, of which 15% are thought to CSNB Microarray be disease causing in DNA samples with previously unknown genotype. In addition to the putative polymorphisms mentioned herein, other sequence variants, probably also representing SNPs or modifiers, can be detected with the CSNB genotyping microar- DISCUSSION ray. A CACNA1F mutation c.2204AϾC (p.Asn735Thr) in exon 16 was has been identified in a patient showing compound In this study we established a mutation detection tool for heterozygous mutations in the CABP4 gene. Since his unaf- CSNB, which overcomes costly, low-sensitivity, and time-con- fected brother showed this substitution also, the sequence suming prescreening methods such as SSCP and DHPLC. Al- variant was classified as a rare polymorphism or modifier8 though direct sequencing is the gold standard for genetic (Tables 3A, B). testing, genetic heterogeneity and large genes containing more Furthermore, three different GRM6 sequence variants were than 30 exons remain labor intensive to investigate. The ad- detected by applying the CSNB microarray (Table 3A): A het- vantage of a CSNB microarray is that this method neither erozygous c.727GϾT transversion (p.Val243Phe) in exon 3 depends on large family pedigrees with more than one patient was originally detected by direct sequencing of GRM6 in a affected nor on a precise clinical discrimination of the different patient with CSNB, in whom the ERG data did not discriminate subforms of CSNB (e.g., incomplete versus complete CSNB; between the complete and incomplete form (patient 13154, Table 4). Tu¨bingen, Germany; CZ, EZ, BW, SK, WB, unpublished data, Initially, mutation analysis in our CSNB cohort was per- 2008). Because of the lack of DNA samples of family members, formed by direct sequencing to cover as many mutations as co-segregation could not be performed. A second mutation was possible on this microarray. By doing so, 21 mutations were not identified. We also detected this variant in another patient identified, including 2 recently published and 7 that had been from Tu¨bingen (7330) showing a nonsense mutation in NYX described earlier. These studies indicated that at least 33% of (p.Gln203Stop). The database GeneCards annotates this GRM6 patients with CSNB carry a known mutation in one of the variant as a rare SNP (rs17078894). An investigation of 172 Eu- known CSNB-associated genes. These findings led to the as- ropeans revealed an allele frequency of G: 0.99, T: 0.01 (No- sumption that a CSNB microarray is a valuable diagnostic tool vember 4, 2008). Two of 178 control alleles analyzed by Dryja for new patients with CSNB. In total of 126 sequence variants et al.29 showed the same variant, suggesting that the GRM6 can be detected by the CSNB microarray. Based on the litera- variant is not disease causing. In patient 8798, a previously ture and our own validations 118 of those are disease causing, unreported c.824GϾA nucleotide exchange (p.Gly275Asp) whereas 8 of them are of uncertain pathogenic character, was identified in exon 3 of GRM6. Direct sequencing of the representing SNPs or modifiers. The microarray was 100% coding exons and flanking intronic regions revealed no second effective in detecting known variants, and 37 known variations mutation, and thus it is not clear whether the c.824GϾA in 39 DNA samples were reliably detected from both strands. exchange is pathogenic. A second mutation may represent a By applying DNA samples from a CSNB cohort with unknown deletion of one or more exons, which would not be detected gene defect, the chip revealed a sequence variant in 18%, of by direct sequencing. The c.824GϾA nucleotide exchange was which 15% are thought to be pathogenic. The detection rate neither published nor predicted as an SNP in the available may change in the future, when more laboratories are aware of databases. Family members were not available for co-segrega- such an array and will analyze their CSNB cases with this tion analyses. Two bioinformatic algorithms, Polyphen and relatively inexpensive screening method. At this time our co- SIFT, were applied to predict the pathogenic character of this hort of patients with CSNB with unknown gene defect was substitution: Polyphen classified this variant as probably dam- small (n ϭ 34). The remaining mutations not detectable by the IOVS, December 2009, Vol. 50, No. 12 CSNB-Genotyping Microarray 5925

TABLE 4. Advantages and Disadvantages of the CSNB Microarray

Advantage Disadvantage

Robust Detects only known variants Can be updated regularly with new mutations Detection rate at the moment only 15%–18%, new mutations can be added only after direct sequencing of CSNB genes Validated with patients from different ethnic backgrounds Number of patients at the moment low (39 DNA samples with known mutation, 34 with unknown genotype in which 6 showed a mutation on microarray screening) Inexpensive If no mutation is detected CSNB genes need to be sequenced directly Simplex cases can be used To conﬁrm pathogenic character of mutation larger family still advantageous for cosegregation studies Mutation detection does not depend on precise clinical discrimination Exclusion of CSNB mutation to use DNA for linkage or candidate gene approaches to identify new genes Prescreening method for diagnostics Sequence validation required Each sequence variant on the microarray can be followed up by Needs careful interpretation and validation of the original reference the given reference

chip may be identified by direct sequencing of known CSNB retinal and optic atrophy with a clinical progressive course of genes or in novel genes underlying this disorder. These data visual dysfunction and to X-linked cone–rod dystrophy.4–7,31 will be then used to update the chip and will result in a higher Similar phenotypic variations have been reported in pa- detection rate in the future. tients carrying CABP4 mutations. Two male patients from the Nevertheless, these initial studies already suggest that the same family with the same homozygous frameshift mutation CSNB microarray is an efficient first-pass screening to detect developed either incomplete CSNB or a more progressive form known variants. It is especially useful for simplex cases, in associated with a decrease in visual acuity and photophobia, patients in whom the mode of inheritance is unclear and in respectively.8 Of interest, just recently a novel homozygous whom the ophthalmic examinations do not discriminate be- mutation in CABP4 (p.Arg216Stop) was described leading to a tween the incomplete and the complete forms of CSNB. It can congenital cone–rod synaptic disorder. The Dutch sib pair also be used to exclude CSNB cases of known mutations to carrying this novel mutation showed reduced visual acuity, furthermore use these samples to identify novel genes under- photophobia, and abnormal color vision, without symptoms of lying CSNB by candidate gene approaches and in larger families night blindness. Clinical presentations and ERG measurements by linkage analysis. Certainly, one must be aware that novel displayed a predominant cone dysfunction.32 mutations in the known genes are missed by this strategy Mutations in CACNA2D4 have been identified in a patient (Table 4). with the full-field ERG results suggestive of incomplete CSNB. Of note, our preliminary screening of CSNB patients with However, the patient showed a mild form of cone dystrophy unknown gene defects revealed six CACNA1F variants (five with a progressive decrease in visual acuity.9 These studies pathogenic and one probable polymorphism or modifier). indicate that the initial clinical diagnosis, especially for incom- CACNA1F consists of 48 coding exons, and thus direct se- plete CSNB, must be validated over a certain time period. In quencing, although it is the gold standard, is still time consum- some cases, the disease course turns out to be progressive ing and costly compared with chip analysis. Therefore, we rather than stationary or can even result in another severe suggest that the chip is particularly useful for patients with the retinal disease. incomplete form of CSNB and in particular for patients with Thus, the CSNB microarray can also be considered for X-linked inheritance. Taken into account that the autosomal patients showing a more progressive form than the classic recessive genes CABP4, CACNA2D4, and GRM6 have been incomplete CSNB. It would also be interesting to investigate only recently associated with CSNB, only a few mutations have whether CACNA1F plays an important role in patients showing been discovered in these genes. Thus, in the near future com- cone or cone–rod dystrophies. prehensive analysis of these genes in families with an autoso- As in our case, screening patients with known disease- mal recessive inheritance may be more successful in identify- associated sequence variations on such a microarray can also ing additional disease-causing mutations. The corresponding reveal unexpected findings. The chip outcome should always oligonucleotides of the newly identified mutations will then be be compared to the respective references presented herein. In added on the array and will make this tool also more attractive addition, for diagnostic purposes, it is mandatory to validate for recessive forms. the outcome by direct sequencing and to perform co-segrega- Furthermore, the CSNB microarray may also be used as a tion analysis if family members are available and, for autosomal prescreening method for patients with more progressive reti- recessive conditions, to screen the whole gene in case of nal disorders including cone- or cone-rod dystrophies. Muta- identification of only one disease allele by the chip. tions in CACNA1F, CABP4, as well as CACNA2D4 have been In conclusion, the microarray presented herein offers a identified in patients initially diagnosed with nonprogressive prescreening tool for CSNB diagnostics. It is not only a cost- CSNB. However, in some cases the phenotype turned out to be efficient method of screening patients with the different forms more progressive than originally believed. The diagnosis is of CSNB but can also be used to test the hypothesis that often based on a first examination by ERG, revealing the typical CACNA1F plays an important role in more progressive retinal electronegative ERG that is associated with CSNB.30 Several disorders like cone- or cone–rod dystrophies. Furthermore, as cases with CACNA1F mutations have been reported in which new mutations are identified, updated versions of the microar- either the same or different mutations lead to different pheno- ray will be generated in regular time intervals. The detailed typic manifestations varying from classical incomplete CSNB to information concerning the origin and clinical context of the 5926 Zeitz et al. IOVS, December 2009, Vol. 50, No. 12 mutations described herein will help to better interpret the 16. Ramensky V, Bork P, Sunyaev S. Human non-synonymous SNPs: results of chip screening. server and survey. Nucleic Acids Res. 2002;30:3894–3900. 17. Ng PC, Henikoff S. Predicting deleterious amino acid substitutions. Acknowledgments Genome Res. 2001;11:863–874. 18. Wutz K, Sauer C, Zrenner E, et al. Thirty distinct CACNA1F muta- The authors thank patients and family members for their contribution; tions in 33 families with incomplete type of XLCSNB and Cacna1f Alfred J. L. G. Pinckers (Ophthalmology, Nijmegen) for ascertaining the expression profiling in mouse retina. Eur J Hum Genet. 2002;10: patients with CSNB; and Markus Preising for the DNA extraction from 449–456. the CSNB samples collected at the Department of Pediatric Ophthal- 19. Leifert D, Todorova MG, Prunte C, Palmowski-Wolfe AM. LED- mology, Strabismology and Ophthalmo-Genetics, University Clinic, Re- generated multifocal ERG on- and off-responses in complete con- gensburg, and at the Department of Ophthalmology, Justus-Liebig- genital stationary night blindness: a case report. Doc Ophthalmol. University Giessen, Universitaetsklinikum Giessen and Marburg GmbH, 2005;111:1–6. Giessen Campus, Giessen, Germany. 20. Zeitz C, Forster U, Neidhardt J, et al. Night blindness-associated mutations in the ligand-binding, cysteine-rich, and intracellular References domains of the metabotropic glutamate receptor 6 abolish protein trafficking. Hum Mutat. 2007;28:771–780. 1. Zeitz C, Gross AK, Leifert D, et al. Identification and functional 21. Boycott KM, Maybaum TA, Naylor MJ, et al. A summary of 20 characterization of a novel rhodopsin mutation associated with CACNA1F mutations identified in 36 families with incomplete autosomal dominant CSNB. 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